Cytotoxicity by natural killer (NK) cells can be inhibited by the expression of class I major histocompatibility (MHC) on target cells. In mice, the class I antigen H2dd specifically binds to the NK antigen Ly-49a and inhibits lysis by the subset of NK cells that express Ly-49a. The epitopes on H2dd that are recognized by Ly-49a have not been defined, but several possibilities exist including; processed peptides, oligosaccharide or the polypeptide backbone of H2dd. My preliminary result indicate that a region in the alpha1 helix of class I heavy chain is involved in the recognition of H2dd by Ly-49a. I find that a soluble chimeric form of Ly-49a can specifically bind a synthetic 12 amino acid peptide that constitutes one portion of the alpha1 helix. In this grant, I propose to define the critical residues and determine the kd of the association. The functional significance of this binding will be assessed by the ability of peptides to both interfere with a cell-cell binding assay and to restore the lysis of Dd expressing targets by Ly-49a+NK cells. Additionally, I plan to expand these findings to the other Ly-49 family members. The critical residues involved in this interaction will determined using a panel of mutated synthetic peptides. Two approached will be employed to assess the peptides ability to bind chimeric Ly-49a; an EIA and the BIAcore real time plasmon resonance machine. One advantage of the BIAcore machine is the ability to calculate the Kd of the binding. These experiment will establish the methodology to study the possible binding of other Ly-49 family members to different MHC class I antigens. Chimeric molecules of these other members will be made and the BIAcore machine used to examine their ability to bind other alpha1 helix peptides. These studies will help define the role of class I antigens in the regulation of NK cell cytotoxicity, one of the most important issues in the field.